I have been able to refold several variants of a 62 residue
b-sheet protein by a 10fold dilution of the protein originally in 6M
urea. I slowly poured the protein solution into a 10 times bigger volume
of refolding buffer cooled on ice and under agitation. The idea is that
the protein gets quickly into a refolding buffer and collapses and refolds
without minimal chances of aggregating with other partially folded
molecules. I don't know if it works in this way, but it worked in my
case. The problem is that you may end up with a big volume of a dilute
protein solution in 0.6M urea. This can be efficiently concentrated by
ultrafiltration in a stirred cell under nitrogen or air pressure (from
Amicon, for example). After that a dialysis against the appropriate
buffer will eliminate the remaining urea.
Good luck
-----------------------------------------------------------------------
Francisco Blanco phone: 301 402 4687
National Institutes of Health fax: 301 496 0825
Laboratory of Chemical Physics, NIDDK blanco@speck.niddk.nih.gov
9000 Rockville Pike, Building 5, room 406
Bethesda, MD 20892 0520, USA
On Wed, 24 Feb 1999, david andreu wrote:
> Hi all:
> We have synthesized antifreeze protein III (sequence below), which in
> its native state belongs to the beta-sandwhich structural group. Our
> synthetic sequence is OK by the usual criteria but we don't seem to be
> able to get it to fold properly; i.e., dialysis of a denatured (6 M
> GdCl) sample against increasingly dilute GdCl doesn't give a product
> with the same CD reported for the native version. Any suggestions,
> tricks, etc?
>
> NQASVVANQL IPINTALTLV MMRSEVVTPV GIPAEDIPRL VSMQVNRAVP
> LGTTLMPDMV KGYAA
>
> Thanks in advance and best regards,
>
> David Andreu
>
> --
> David Andreu
> University of Barcelona, Spain
> Phone/Fax: 34-934 021 260
> andreu@admin.qo.ub.es
>
>
>